Globo H and related anti-cancer vaccines with novel glycolipid adjuvants

ABSTRACT

An immunogenic composition containing a glycan conjugate including a carrier protein, and a glycan including Globo H, an immunogenic fragment thereof, or stage-specific embryonic antigen-4 (SSEA-4), wherein the glycan is conjugated with the carrier protein through a linker.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation application of U.S. patentapplication Ser. No. 13/568,510, filed on Aug. 7, 2012, which is acontinuation application of U.S. patent application Ser. No. 12/537,129,filed on Aug. 6, 2009, now issued as U.S. Pat. No. 8,268,969 and isrelated to U.S. patent application Ser. No. 12/485,546, filed Jun. 16,2009, now abandoned, which claims priority to U.S. Provisional PatentApplication No. 61/061,968 filed Jun. 16, 2008. The contents of thesepatent applications are incorporated herein in their entirety byreference.

TECHNICAL FIELD OF THE INVENTION

This invention relates to the field of cancer vaccines. In particular,the application relates to a carbohydrate-based vaccine containing the Bcell epitope, Globo H, which is conjugated to the immunogenic carrierDT-CRM197. More particularly, the invention is directed at anti-cancerGlobo H-DT vaccines administered with novel glycolipid adjuvants, suchas C34.

BACKGROUND OF THE INVENTION

To design therapy against cancer, it is desirable to seek moleculartargets of cancer or cancer stem cells that are absent from normalcells. Aberrant glycosylation is often associated with tumor progressionand was first described by Meezan et al. in 1969 with the demonstrationthat cancer glycans differ from healthy cells. (Meezan E, et al. (1969)Biochemistry 8:2518-2524.) Aberrant glycosylations include loss orover-expression of certain structures, the persistence of truncatedstructures and the emergence of novel structures. The structuraldifferences were later supported by many histological evidences usinglectin-staining compared with healthy and malignant tissue. (Turner G A(1992) Clin Chim Acta 208:149-171; Gabius H J (2000) Naturwissenschaften87:108-121.)

More recently, tumor associated carbohydrate antigens were identified bymonoclonal antibodies and mass spectrometry. (Shriver Z, et al. (2004)Nat Rev Drug Disc 3:863-873; Pacino G, et al. (1991) Br J Cancer63:390-398.) To date, numerous tumor associated antigens expressed oncancer cells in the form of glycolipids or glycoproteins have beencharacterized and correlated to certain types of cancers. (Bertozzi C R,Dube D H (2005) Nat Rev Drug Discovery 4:477-488.) Although relativelylittle is known about the role of surface carbohydrates play inmalignant cells, passively administered or vaccine induced antibodiesagainst these antigens have correlated with improved prognosis.

Of the tumor associated glycans reported, the glycolipid antigen Globo H(Fucα1→2 Galβ→3 GalNAcβ1→3 Galα1→4 Galβ→4 Glc) was first isolated andidentified in 1984 by Hakomori et al. from breast cancer MCF-7 cells.(Bremer E G, et al. (1984) J Biol Chem 259:14773-14777.) Further studieswith anti-Globo H monoclonal antibodies showed that Globo H was presenton many other cancers, including prostate, gastric, pancreatic, lung,ovarian and colon cancers and only minimal expression on luminal surfaceof normal secretory tissue which is not readily accessible to immunesystem. (Ragupathi G, et al. (1997) Angew Chem Int Ed 36:125-128.) Inaddition, it has been established that the serum of breast cancerpatient contains high level of anti-Globo H antibody. (Gilewski T el al.(2001) Proc Natl Acad Sci USA 98:3270-3275; Huang C-Y, et al. (2006)Proc Natl Acad Sci USA 103:15-20; Wang C-C, et al. (2008) Proc Natl AcadSci USA 105(33):11661-11666) and patients with Globo H-positive tumorsshowed a shorter survival in comparison to patients with GloboH-negative tumors. (Chang, Y-J, et al. (2007) Proc Natl Acad Sci USA104(25):10299-10304.) These findings render Globo H, a hexasaccharideepitope, an attractive tumor marker and a feasible target for cancervaccine development.

Globo H is a cancer antigen overly expressed in various epithelialcancers. It has been suggested that this antigen can serve as a targetin cancer immunotherapy. While vaccines have been developed to elicitantibody responses against Globo H, their anti-cancer efficacies areunsatisfactory due to low antigenicity of Globo H. There is a need for anew vaccine capable of eliciting high levels of immune responsestargeting Globo H.

Stem cells are defined as a group of cells with the capacity forself-renewal and for differentiation into different types of cells andtissues. (Reya T et al., (2001) Nature 414:105-111.) As both malignanttumors and normal tissues contain heterogeneous populations of cells,cancer stem cells might play a key role in tumor growth and maintainingtumor heterogeneity. Cancer stem cells have been identified from avariety of solid tumors, such as brain, breast, colon, and prostatecancers. Breast cancer stem cells (BCSCs) were first shown to reside inthe CD24⁻CD44⁺ subpopulation of breast cancer by Al-Hajj et al., basedon their ability to generate tumors with phenotypic diversity onxenotransplantation into NOD/SCID mice (Al-Hajj M, et al., (2003) ProcNatl Acad Sci USA 100:3983-3988). The majority of early disseminatedcancer cells in the bone marrow of breast cancer patients displayed thephenotype of CD24⁻CD44⁺ (Balic Metal., (2006) Clin Cancer Res12:5615-5621), suggesting that BCSCs were capable of metastasis. Basedon their capability for growth, differentiation, and metastasis andtheir resistance to radiation, BCSCs are a major target for therapy ofbreast cancer (Tang C. et al., (2007) FASEB J. 21:1-9).

In breast cancer, Globo H expression was observed in >60% of ductal,lobular, and tubular carcinoma, but not in nonepithelial breast tumors(Mariani-Constantini R et al., (1984) Am. J. Pathol. 115:47-56). Globo His not expressed in normal tissue except for weak expression in theapical epithelial cells at lumen borders, a site that appears to beinaccessible to the immune system (Id.; Zhang S. et al., (1997) Int. J.Cancer 73:42-49).

Globo H also is expressed in breast cancer stem cells (BCSCs). Flowcytometry revealed Globo H is expressed in 25/41 breast cancer specimens(61.0%). Non-BCSCs from 25/25 and BCSCs from 8/40 (20%) express Globo H.The stage-specific embryonic antigen 3 (SSEA-3), the pentasaccharideprecursor of Globo H, is expressed in 31/40 (77.5%) tumors. Non-BCSCsfrom 29/31 and BCSCs from 25/40 (62.5%) expressed SSEA-3. (Chang W-W. etal., (2008) Proc Natl Acad Sci USA 105(33):11667-11672.)

Danishefsky and Livingston previously reported the preparation of GloboH-KLH vaccine (Gilewski T el al. (2001) Proc Natl Acad Sci USA98:3270-3275; Ragupathi G, et al. (1997) Angew Chem Int Ed 36:125-128;Kudryashov V, et al. (1998) Glycoconj J. 15:243-249; Slovin S F et at(1997) Proc Natl Acad Sci USA 96:5710-5715) and the heptavalent vaccine(containing GM2, Globo H, Lewis Y, Tn, STn, TF, and Tn-MUC1 individuallyconjugated to KLH; Sabbatini P J et at (2007) Clin Cancer Res13:4170-4177) against a variety of cancers. However, patients immunizedwith the heptavalent vaccine induced antibody responses against onlyfive of the seven antigens except GM2 and Lewis Y antibodies. Ratherthan ubiquitously expressed antigen such as GM2, Globo H exceptionallyexpressed on tumor cells with only minimal level on normal secretorytissue makes it a desirable target for vaccine development. In theirstudies, ozonolysis of Globo H aglycone was followed by reductiveamination with KLH carrier protein to generate about 150 carbohydrateunits per protein. (Ragupathi G, et al. (1997) Angew Chem Int Ed36:125-128.) Further refinement increased the carbohydrate conjugationratio to about 720:1 by using MMCCH linker. (Wang S-K, et al. (2008).Proc Natl Acad Sci USA 105:3690-3695.) However, it was difficult toprecisely characterize the glycoconjugate. In addition, the syntheticvaccine in combination with the immunological adjuvant QS-21 was shownto induce mainly IgM and to a lesser extent IgG antibodies in bothprostate and metastatic breast cancer patients. In the phase I clinicaltrial, the vaccine also showed minimal toxicity with transient localskin reactions at the vaccination site. (Gilewski T el al. (2001) ProcNatl Acad Sci USA 98:3270-3275; Ragupathi G, et al. (1997) Angew ChemInt Ed 36:125-128; Slovin S F et at (1997) Proc Natl Acad Sci USA96:5710-5715.) Mild flu-like symptoms which have been observed in someof the patients were probably associated with the side effect of QS-21.A pentavalent vaccine containing five prostate and breast cancerassociated carbohydrate antigens—Globo-H, GM2, STn, TF and Tn—conjugatedto maleimide-modified carrier protein KLH has been reported to produceanti-Globo H sera with higher titers of IgG than IgM in ELISA assays.(Zhu J. et al. (2009) J. Am. Chem. Soc. 131(26):9298-9303).

Therefore, it is desirable to identify an alternative carrier andadjuvant to augment the antibody response to Globo H, especially withhigh titer of IgG, and to improve the vaccine efficacy with minimal sideeffects.

SUMMARY OF THE INVENTION

This invention relates to a carbohydrate based vaccine containing GloboH (B cell epitope) chemically conjugated to the immunogenic carrierdiphtheria toxin cross-reacting material 197 (DT-CRM197) (Th epitope)via a p-nitrophenyl linker. The synthetic vaccine in combination with aglycolipid adjuvant induce IgG, IgG1 and IgM antibodies and provided anexceptional immunogenicity in breast cancer models, showing delayedtumorigenesis in xenograft studies. Glycan array analysis of theantibodies induced by Globo H-DT and the glycolipid C34 showed that theantibodies not only recognized Globo H but also SSEA-3 (Gb5) and SSEA-4(sialyl Gb5) glycans, all specific for cancer cells and cancer stemcells.

The invention relates to an immunogenic composition comprising: (a) aglycan consisting essentially of Globo H or an immunogenic fragmentthereof, wherein the glycan is conjugated with a carrier protein througha linker; and (b) an adjuvant comprising a glycolipid capable of bindinga CD1d molecule on a dendritic cell, wherein the immunogenic compositioninduces an immune response that induces a higher relative level of IgGisotype antibodies as compared to IgM isotype antibodies.

In some aspects, the carrier protein is diphtheria toxin cross-reactingmaterial 197 (DT-CRM197). In some aspects, the linker is a p-nitrophenyllinker.

In some aspects, the adjuvant is a synthetic analog ofα-galactosyl-ceramide (α-GalCer). In some embodiments the adjuvant isC34, wherein C34 comprises the structure:

In some aspects, the immune response is preferably oriented towards theproduction of IgG isotype antibodies. In some aspects, the immunogeniccomposition comprises at least one adjuvant able to induce a humoral andcellular immune response.

In some aspects, the antibodies generated by the immune responseneutralize antigens expressed on cancer cells or cancer stem cells. Insome embodiments, the antibodies generated by the immune responseneutralize at least one of the antigens Gb4, stage-specific embryonicantigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4). Insome embodiments, the antibodies that neutralize at least one of theantigens Gb4, stage-specific embryonic antigen-3 (SSEA-3) andstage-specific embryonic antigen-4 (SSEA-4) comprise a higher relativelevel of IgG isotype antibodies as compared to IgM isotype antibodies.

The invention relates to a cancer vaccine comprising the immunogeniccomposition which is able to induce anti-cancer immune responses in asubject. In some aspects, the cancer vaccine is suitable for treating acancer selected from the group consisting of: breast cancer, lungcancer, liver cancer, buccal cancer, stomach cancer, colon cancer,nasopharyngeal cancer, dermal cancer, renal cancer, brain tumor,prostate cancer, ovarian cancer, cervical cancer, intestinal cancer, andbladder cancer.

In some aspects, the cancer tissue expresses a Globo H antigen on thesurface of the cell. In some aspects, the Globo H antigen is expressedon an epithelial cell of a breast tumor.

In some embodiments, the cancer vaccine generates antibodies thatneutralize at least one of the antigens Globo H, Gb4, stage-specificembryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4(SSEA-4). In some aspects, the antigens are expressed on a breast cancerstem cell.

The invention relates to a method of treatment comprising inhibition oftumor growth, the method comprising: (a) administering to a subject inneed thereof, an immunogenic composition comprising: a glycan consistingessentially of Globo H or an immunogenic fragment thereof, wherein theglycan is conjugated with a carrier protein through a linker, and anadjuvant comprising a glycolipid capable of binding a CD1d molecule on adendritic cell; and (b) inducing an immune response that induces ahigher relative amount of IgG isotype antibodies as compared to IgMisotype antibodies.

In some embodiments of the method, the linker is p-nitrophenol, thecarrier protein is diphtheria toxin cross-reacting material 197(DT-CRM197) and the adjuvant is a synthetic analog ofα-galactosyl-ceramide (α-GalCer). In one embodiment, the adjuvant isC34.

In some embodiments of the method, the immunogenic composition furthercomprises a cancer vaccine, and further wherein one or more treatmentswith an effective amount of the cancer vaccine inhibits tumor growth. Insome embodiments, administration of the cancer vaccine reduces the sizeof a tumor.

In some embodiments of the method, wherein the immune response ispreferably oriented towards the production of IgG isotype antibodiesthat neutralize at least one of the antigens Globo H, Gb4,stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonicantigen-4 (SSEA-4). In some aspects, at least one of the antigens GloboH, stage-specific embryonic antigen-3 (SSEA-3) and stage-specificembryonic antigen-4 (SSEA-4) is expressed on a breast cancer stem cell.In some aspects, the Globo H antigen is expressed on an epithelial cellof a breast tumor.

The invention relates to a cancer vaccine comprising: (a) an immunogeniccomposition comprising: a glycan consisting essentially of Globo H or animmunogenic fragment thereof, wherein the glycan is conjugated with acarrier protein through a linker, and an adjuvant comprising aglycolipid capable of binding a CD1d molecule on a dendritic cell,wherein the immunogenic composition induces an immune response thatinduces a higher relative level of IgG isotype antibodies as compared toIgM isotype antibodies; and (b) a pharmaceutically acceptable excipient.

In some aspects, the cancer vaccine comprises an immunogenic compositionthe linker is p-nitrophenol, the carrier protein is diphtheria toxincross-reacting material 197 (DT-CRM197) and the adjuvant is a syntheticanalog of α-galactosyl-ceramide (α-GalCer). In one embodiment, theadjuvant is C34.

In some aspects, the cancer vaccine is used to treat a cancer, whereinone or more treatments with an effective amount of the cancer vaccineinhibits tumor growth. In some embodiments, administration of the cancervaccine reduces the size of a tumor. In some embodiments, the cancer isselected from the group consisting of: breast cancer, lung cancer, livercancer, buccal cancer, stomach cancer, colon cancer, nasopharyngealcancer, dermal cancer, renal cancer, brain tumor, prostate cancer,ovarian cancer, cervical cancer, intestinal cancer, and bladder cancer.

The invention relates to an immunogenic composition comprising: (a) aglycan consisting essentially of a Globo H-related glycan or animmunogenic fragment thereof, wherein the glycan is conjugated with acarrier protein through a linker; and (b) an adjuvant comprising aglycolipid capable of binding a CD1d molecule on a dendritic cell,wherein the Globo H-related glycan is selected from the group consistingof SSEA-3 and SSEA-4, and wherein the immunogenic composition induces animmune response that induces a higher relative level of IgG isotypeantibodies as compared to IgM isotype antibodies.

In some aspects of the immunogenic composition the carrier protein isdiphtheria toxin cross-reacting material 197 (DT-CRM197), the adjuvantis a synthetic analog of α-galactosyl-ceramide (α-GalCer) and the linkeris a p-nitrophenyl linker. In one embodiment, the adjuvant is C34

The invention relates to a therapeutic against breast cancer stem cells,the therapeutic comprising: Globo H conjugated through a p-nitrophenyllinker with a diphtheria toxin cross-reacting material 197 (DT-CRM197)carrier protein; and an adjuvant comprising a glycolipid capable ofbinding a CD1d molecule on a dendritic cell. In some embodiments of thetherapeutic, the adjuvant is C34.

The invention relates to a therapeutic against breast cancer stem cells,the therapeutic comprising: SSEA-3 conjugated through a p-nitrophenyllinker with a diphtheria toxin cross-reacting material 197 (DT-CRM197)carrier protein; and an adjuvant comprising a glycolipid C34 capable ofbinding a CD1d molecule on a dendritic cell.

The invention relates to a therapeutic against breast cancer stem cells,the therapeutic comprising: SSEA-4 conjugated through a p-nitrophenyllinker with a diphtheria toxin cross-reacting material 197 (DT-CRM197)carrier protein. In some embodiments, the therapeutic further comprisesan adjuvant comprising a glycolipid capable of binding a CD1d moleculeon a dendritic cell.

Administration of the therapeutics of the invention to a subject inducesproduction of antibodies that recognize an antigen expressed on a breastcancer stem cell (BCSC), wherein the antigen is selected from the groupconsisting of Globo H, SSEA-3 and SSEA-4. The invention relates to amethod of treating breast cancer comprising administration of atherapeutic of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, the inventions of which can be better understood byreference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein. Thepatent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows structures of Globo H and truncated derivatives.

FIGS. 2A-2C show binding specificity of monoclonal antibodies VK9 andMbr1 (to Globo H) and anti-SSEA-3, respectively.

FIGS. 3A-3B show serologic response of mice vaccinated with variousGlobo H conjugates and α-GalCer. Groups of three C57BL/6 mice werevaccinated s.c. with 1 μg of synthetic glycoconjugates with or without 2μg glycolipid. Mice sera were diluted 1:60 and 1:240 respectively forIgM (FIG. 3A) and IgG (FIG. 3B) antibody analysis. Cy3-anti-mouse IgG orIgM secondary antibodies were used for fluorescence detection under 532nm, PMT 500, Data represent as average fluorescence intensity of threemice±the SEM.

FIG. 4 shows structures of α-GalCer and analogues

FIG. 5 shows IgM levels of mice vaccinated with Globo H conjugates andα-GalCer derivatives. Mouse sera were collected and analyzed after2^(nd) and 3^(rd) vaccinations, as shown. Cy3 secondary anti-mouse IgMwas used for detection under 532 nm, PMT 400. The results representaverage fluorescence intensity of three mice±the SEM.

FIG. 6 shows fine specificity of mouse polyclonal antibody (anti-GloboH, anti-Gb5, anti-SSEA-4 and anti-Gb4) after vaccination. Mice sera wereobtained two weeks after the 3^(rd) vaccination of 1.6 μg GH-DT with orwithout 2 μg of adjuvant. (Female, Balb/c, i.m.) The IgG titers wereanalyzed by glycan microarray and defined as the highest dilutionyielding the MFI greater than 1000 (10 folds over background), PMT 400.Each spot presents as individual mouse titer.

FIG. 7 shows IgM vs IgG antibody titers of Globo H-DT with differentadjuvants.

FIG. 8 shows evaluation of the adjuvant activities with GH-KLH vaccines.Female Balb/c mice were vaccinated i.m. with 1.6 μg GH-KLH and 2 μgindicated adjuvants and bled every two weeks after vaccination. The serawere diluted and introduced to microarray analysis.

FIG. 9 shows antibody isotype profile after immunizations. Mice werevaccinated as described. Sera (1:60 dilutions) were introduced tomicroarray for antibody subclasses analysis (532 nm, PMT 300). Datapresents as mean fluorescence of three mice±the SEM.

FIG. 10 shows antibody titers of IgM vs IgG induced by SSEA-3-DT orSSEA-4-DT with different kind of glycolipid adjuvants.

FIG. 11 shows structures of 24 glycans on the cell surface.

FIGS. 12A-12C show cross-reactivity studies of induced IgG by differentvaccines. FIG. 12 A: anti-Globo H IgG induced by Globo H-DT with C1adjuvant; FIG. 12 B: anti-Gb5 IgG induced by Gb5-DT with C1 adjuvant;FIG. 12 C: anti-SSEA-4 IgG induced by SSEA-4-DT with C1 adjuvant.

FIG. 13 shows a mouse xenograft model. 2×10⁵ 4 T1 mouse metastaticmammary tumor cells were prepared in sterile PBS and injectedsubcutaneously to vaccinated Balb/c mice. Mouse tumor size was measuredby Vernier caliper and defined as (length×width×width)/2 (mm³)

FIG. 14 shows a schematic for the synthesis of Globo H half ester andglycoconjugates.

FIG. 15 shows flow cytometric analysis of SSEA-4 expression in primarybreast cancer stem cells. Expression of SSEA-4 on the surface of BCSCsand non-BCSCs was evaluated with four-color immunofluorescence stainingand subsequent flow cytometric analysis. BCSCs were defined asCD45⁻/CD24⁻/CD44⁺ cells, and non-BCSCs were defined as other populationsof CD45⁻ cells, as shown in left panel. Expression of antigens ofinterest on BCSCs and non-BCSCs is shown in the middle and right panel,respectively. The dotted line represents isotype control, and thenumbers represent the percentage of positive cells.

FIG. 16 shows restricted expression of SSEA-4 in normal tissues.Immunohistochemical staining of normal tissue arrays was used to examinethe expression of SSEA-4 in breast, small intestine, and rectum.Positive staining for SSEA-4 was restricted to the apical surface ofepithelial cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the surprising finding that DT-CRM197is a promising carrier protein for Globo H and SSEA-4 not only becauseit has been widely used for human vaccination against diphtheria fordecades, but also because of its highly immunogenic property. Mostimportantly, it has been approved by the FDA for various glycoconjugatevaccines. Diphtheria toxin cross-reacting material 197 (DT-CRM197) is anontoxic mutant (G52E) of DT that shares the immunological properties ofthe native molecule and its ability to bind to heparin-binding,epidermal growth factor (HB-EGF), the specific cell-membrane receptorfor DT that is often overexpressed in cancer. (Buzzi S. et al., CancerImmunology, Immunotherapy (2004), 53(11):1041-1048).

Using C34 as adjuvant, both GH-DT and SSEA-4-DT showed the mosteffective immune response to induce more IgG than IgM antibodies againsttumor antigens. The GH-DT in combination with C34 induced antibodieswhich not only neutralize Globo H but also SSEA-3 (Gb5) and SSEA-4,which all are specific for breast cancer cells and the cancer stemcells.

Further, the disclosed glycan microarray offers a powerful platform forantibody specificity test and is useful for identification of patientsfor the vaccine trial and for the monitoring of their immune responseafter immunization.

In the following description, reference is made to the accompanyingdrawings that form a part hereof, and in which is shown by way ofillustration specific embodiments which may be practiced. Theseembodiments are described in detail to enable those skilled in the artto practice the invention, and it is to be understood that otherembodiments may be utilized and that structural, logical and electricalchanges may be made without departing from the scope of the presentinvention. The following description of example embodiments is,therefore, not to be taken in a limited sense, and the scope of thepresent invention is defined by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, cell lines,vectors, animals, instruments, statistical analysis and methodologieswhich are reported in the publications which might be used in connectionwith the invention. All references cited in this specification are to betaken as indicative of the level of skill in the art. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

DEFINITIONS

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes(IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning(1984); the treatise, Methods In Enzymology (Academic Press, Inc.,N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P.Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology,Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Antibodies: A Laboratory Manual, by Harlow and Lane s (ColdSpring Harbor Laboratory Press, 1988); and Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).

As used herein, the term “lipid” refers to any fat-soluble (lipophilic)molecule that participates in cell signaling pathways.

As used herein, the term “glycolipid” refers to a carbohydrate-attachedlipid that serves as a marker for cellular recognition.

As used herein, the term “alpha-galactosyl ceramide” and “α-GalCer”refers to a glycolipid that stimulates natural killer T cells to produceboth T helper (TH)1 and TH2 cytokines. As used herein, the glycolipidderivative C34 has the following structure:

The α-GalCer analogs of the present disclosure include α-GalCer analogsof bacterial origin (Group I: C2, C3 and C14), α-GalCer analogs modifiedwith sulfonation (Group II: C4, C5 and C9), phenyl-alkyl chain α-GalCeranalogs (Group III: C6-C8, C10-C11, C15-C16, C18-C34, C8-5 and C8-6) andphytosphingosine truncated α-GalCer analogs (Group IV: C12, C13 andC17). The structures of C34 and other alpha-galactosyl ceramide analogsand their use as adjuvants are disclosed in detail in PCT patentApplication No. PCT/US2008/060275 filed Apr. 14, 2008.

The synthetic α-GalCer analogs, including C34, are capable of formingcomplexes with a CD1d molecule. Synthetic α-GalCer analogs are capableof being recognized by NKTs T-cell receptors. Synthetic α-GalCer analogsare capable of eliciting a T_(H)1-type, a T_(H)2-type or a TH1-type anda TH2-type response. The α-GalCer analogs are capable of activating NKTsin vitro. α-GalCer analogs are capable of activating NKTs in vivo.

As used herein, the term “glycan” refers to a polysaccharide, oroligosaccharide. Glycan is also used herein to refer to the carbohydrateportion of a glycoconjugate, such as a glycoprotein, glycolipid,glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or aproteoglycan. Glycans usually consist solely of O-glycosidic linkagesbetween monosaccharides. For example, cellulose is a glycan (or morespecifically a glucan) composed of β-1,4-linked D-glucose, and chitin isa glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can behomo or heteropolymers of monosaccharide residues, and can be linear orbranched. Glycans can be found attached to proteins as in glycoproteinsand proteoglycans. They are generally found on the exterior surface ofcells. O- and N-linked glycans are very common in eukaryotes but mayalso be found, although less commonly, in prokaryotes. N-Linked glycansare found attached to the R-group nitrogen (N) of asparagine in thesequon. The sequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is anyamino acid except praline.

As used herein, the term “glycoprotein” refers to a protein covalentlymodified with glycan(s). There are four types of glycoproteins: 1)N-linked glycoproteins, 2) O-linked glycoproteins (mucins), 3)glucosaminoglycans (GAGs, which are also called proteoglycans), 4)GPI-anchored. Most glycoproteins have structural micro-heterogeneity(multiple different glycan structures attached within the sameglycosylation site), and structural macro-heterogeneity (multiple sitesand types of glycan attachment).

As used herein, the term “antigen” is defined as any substance capableof eliciting an immune response.

As used herein, the term “immunogen” refers to an antigen or a substancecapable of inducing production of an antigen, such as a DNA vaccine.

As used herein, the term “immunogenicity” refers to the ability of animmunogen, antigen, or vaccine to stimulate an immune response.

As used herein, the term “immunotherapy” refers to an array of treatmentstrategies based upon the concept of modulating the immune system toachieve a prophylactic and/or therapeutic goal.

As used herein, the term “CD1d” refers to a member of the CD1 (clusterof differentiation 1) family of glycoproteins expressed on the surfaceof various human antigen-presenting cells. CD1d presented lipid antigensactivate natural killer T cells. CD1d has a deep antigen-binding grooveinto which glycolipid antigens bind. CD1 d molecules expressed ondendritic cells can bind and present glycolipids, including α-GalCeranalogs such as C34.

As used herein, the term “adaptive immune system” refers to highlyspecialized, systemic cells and processes that eliminate pathogenicchallenges. The cells of the adaptive immune system are a type ofleukocyte, called a lymphocyte. B cells and T cells are the major typesof lymphocytes.

As used herein, the term “T cells” and “Ts” refer to a group of whiteblood cells known as lymphocytes, that play a central role incell-mediated immunity. T cells can be distinguished from otherlymphocyte types, such as B cells and NKs by the presence of a specialreceptor on their cell surface called the T cell receptor (TCR). Severaldifferent subsets of T cells have been described, each with a distinctfunction. Helper T (T_(H)) Cells are the “middlemen” of the adaptiveimmune system. Once activated, they divide rapidly and secrete smallproteins called cytokines that regulate or “help” the immune response.Depending on the cytokine signals received, these cells differentiateinto T_(H)1, T_(H)2, T_(H)17, or one of other subsets, which secretedifferent cytokines.

As used herein, the term “antigen-presenting cell” (APC) refers to acell that displays foreign antigen complexed with majorhistocompatibility complex (MHC) on its surface. T-cells may recognizethis complex using their TCR. APCs fall into two categories:professional or non-professional. Dendritic cells (DCs) fall under theprofessional category and are capable of presenting antigen to T cells,in the context of CD1. In an exemplary implementation, the DCs utilizedin the methods of this disclosure may be of any of several DC subsets,which differentiate from, in one implementation, lymphoid or, in anotherimplementation, myeloid bone marrow progenitors.

As used herein, the term “naïve cell” refers to an undifferentiatedimmune system cell, for example a CD4 T-cell, that has not yetspecialized to recognize a specific pathogen.

As used herein, the term “natural killer cells” and “NKs” refers to aclass of lymphoid cells which are activated by interferons to contributeto innate host defense against viruses and other intracellularpathogens.

As used herein, the term “natural killer T cells” (NKTs) refers to asubset of T cells that share characteristics/receptors with bothconventional Ts and NKs. Many of these cells recognize thenon-polymorphic CD1d molecule, an antigen presenting molecule that bindsself- and foreign lipids and glycolipids. The TCR of the NKTs are ableto recognize glycolipid antigens presented (chaperoned) by a CD1dmolecule. A major response of NKTs is rapid secretion of cytokines,including IL-4, IFN-γ and IL-10 after stimulation and thus influencediverse immune responses and pathogenic processes. The NKTs may be ahomogenous population or a heterogeneous population. In one exemplaryimplementation, the population may be “non-invariant NKTs”, which maycomprise human and mouse bone marrow and human liver T cell populationsthat are, for example, CD1d-reactive non-invariant T cells which expressdiverse TCRs, and which can also produce a large amount of IL-4 andIFN-γ. The best known subset of CD1d-dependent NKTs expresses aninvariant TCR-alpha (TCR-α) chain. These are referred to as type I orinvariant NKTs (iNKTs). These cells are conserved between humans (Vα24iNKTs) and mice (Vα14i NKTs) and are implicated in many immunologicalprocesses.

As used herein, the term “cytokine” refers to any of numerous small,secreted proteins that regulate the intensity and duration of the immuneresponse by affecting immune cells differentiation process usuallyinvolving changes in gene expression by which a precursor cell becomes adistinct specialized cell type. Cytokines have been variously named aslymphokines, interleukins, and chemokines, based on their presumedfunction, cell of secretion, or target of action. For example, somecommon interleukins include, but are not limited to, IL-12, IL-18, IL-2,IFN-γ, TNF, IL-4, IL-10, IL-13, IL-21 and TGF-β.

As used herein, the term “chemokine” refers to any of various smallchemotactic cytokines released at the site of infection that provide ameans for mobilization and activation of lymphocytes. Chemokines attractleukocytes to infection sites. Chemokines have conserved cysteineresidues that allow them to be assigned to four groups. The groups, withrepresentative chemokines, are C—C chemokines (RANTES, MCP-1, MIP-1α,and MIP-1β), C—X—C chemokines (IL-8), C chemokines (Lymphotactin), andCXXXC chemokines (Fractalkine).

As used herein, the term “T_(H)2-type response” refers to a pattern ofcytokine expression such that certain types of cytokines, interferons,chemokines are produced. Typical T_(H)2 cytokines include, but are notlimited to, IL-4, IL-5, IL-6 and IL-10.

As used herein, the term “T_(H)1-type response” refers to a pattern ofcytokine expression such that certain types of cytokines, interferons,chemokines are produced. Typical T_(H)1 cytokines include, but are notlimited to, IL-2, IFN-γ, GMCSF and TNF-β.

As used herein, the term “T_(H)1 biased” refers to am immunogenicresponse in which production of T_(H)1 cytokines and/or chemokines isincreased to a greater extent than production of T_(H)2 cytokines and/orchemokines.

As used herein, the term “epitope” is defined as the parts of an antigenmolecule which contact the antigen binding site of an antibody or a Tcell receptor.

As used herein, the term “vaccine” refers to a preparation that containsan antigen, consisting of whole disease-causing organisms (killed orweakened) or components of such organisms, such as proteins, peptides,or polysaccharides, that is used to confer immunity against the diseasethat the organisms cause. Vaccine preparations can be natural, syntheticor derived by recombinant DNA technology.

As used herein, the term “immunologic adjuvant” refers to a substanceused in conjunction with an immunogen which enhances or modifies theimmune response to the immunogen. The α-GalCer analogs of the presentdisclosure are used as immunologic adjuvants to modify or augment theeffects of a vaccine by stimulating the immune system of a patient whois administered the vaccine to respond to the vaccine more vigorously.In an exemplary implementation, the analog C34 is used as an adjuvant.

As used herein, the term “alum adjuvant” refers to an aluminum salt withimmune adjuvant activity. This agent adsorbs and precipitates proteinantigens in solution; the resulting precipitate improves vaccineimmunogenicity by facilitating the slow release of antigen from thevaccine depot formed at the site of inoculation.

As used herein, the term “anti-tumor immunotherapy active agent” refersto antibody generated by a vaccine of the of the present disclosure thatinhibits, reduces and/or eliminates tumors.

As used herein, the term “antigen specific” refers to a property of acell population such that supply of a particular antigen, or a fragmentof the antigen, results in specific cell proliferation.

As used herein, the term “Flow cytometry” or “FACS” means a techniquefor examining the physical and chemical properties of particles or cellssuspended in a stream of fluid, through optical and electronic detectiondevices.

Amino acid residues in peptides shall hereinafter be abbreviated asfollows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine isIle or I; Methionine is Met or M; Valine is Val or V; Serine is Ser orS; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A;Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q;Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D;Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W;Arginine is Arg or R; and Glycine is Gly or G. For further descriptionof amino acids, please refer to Proteins: Structure and MolecularProperties by Creighton, T. E., W. H. Freeman & Co., New York 1983.

The compositions disclosed herein can be included in a pharmaceutical ornutraceutical composition together with additional active agents,carriers, vehicles, excipients, or auxiliary agents identifiable by aperson skilled in the art upon reading of the present disclosure.

The pharmaceutical or nutraceutical compositions preferably comprise atleast one pharmaceutically acceptable carrier. In such pharmaceuticalcompositions, the compositions disclosed herein form the “activecompound,” also referred to as the “active agent.” As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions. A pharmaceutical composition isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol, or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates, or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

Subject as used herein refers to humans and non-human primates (e.g.,guerilla, macaque, marmoset), livestock animals (e.g., sheep, cow,horse, donkey, and pig), companion animals (e.g., dog, cat), laboratorytest animals (e.g., mouse, rabbit, rat, guinea pig, hamster), captivewild animals (e.g., fox, deer), and any other organisms who can benefitfrom the agents of the present disclosure. There is no limitation on thetype of animal that could benefit from the presently described agents. Asubject regardless of whether it is a human or non-human organism may bereferred to as a patient, individual, animal, host, or recipient.

Pharmaceutical compositions suitable for an injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. It should be stable under theconditions of manufacture and storage and be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying, which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, or adjuvant materials can beincluded as part of the composition. The tablets, pills, capsules,troches and the like can contain any of the following ingredients, orcompounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch orlactose, a disintegrating agent such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate or sterotes; a glidantsuch as colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be transmucosal or transdermal. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration may be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art. The compounds can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

According to implementations, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811, which is incorporated by reference herein.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected location to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the disclosure, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of the activecompound (i.e., an effective dosage) may range from about 0.001 to 100g/kg body weight, or other ranges that would be apparent and understoodby artisans without undue experimentation. The skilled artisan willappreciate that certain factors can influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health or age of the subject, and other diseases present.

According to another aspect, one or more kits of parts can be envisionedby the person skilled in the art, the kits of parts to perform at leastone of the methods herein disclosed, the kit of parts comprising two ormore compositions, the compositions comprising alone or in combinationan effective amount of the compositions disclosed herein according tothe at least one of the above mentioned methods.

The kits possibly include also compositions comprising active agents,identifiers of a biological event, or other compounds identifiable by aperson skilled upon reading of the present disclosure. The kit can alsocomprise at least one composition comprising an effective amount of thecompositions disclosed herein or a cell line. The compositions and thecell line of the kits of parts to be used to perform the at least onemethod herein disclosed according to procedure identifiable by a personskilled in the art.

As used herein, the term “polypeptide” refers to any multimer or polymerof amino acid residues. A polypeptide may be composed of two or morepolypeptide chains. A polypeptide includes a protein, a peptide, and anoligopeptide. A polypeptide can be linear or branched. A polypeptide cancomprise modified amino acid residues, amino acid analogs ornon-naturally occurring amino acid residues and can be interrupted bynon-amino acid residues. Included within the definition are amino acidpolymers that have been modified, whether naturally or by intervention,e.g., formation of a disulfide bond, glycosylation, lipidation,methylation, acetylation, phosphorylation, or by manipulation, such asconjugation with a labeling component.

As used herein, the term “specifically binding,” refers to theinteraction between binding pairs (e.g., an antibody and an antigen). Invarious instances, specifically binding can be embodied by an affinityconstant of about 10⁻⁶ moles/liter, about 10⁻⁷ moles/liter, or about10⁻⁸ moles/liter, or less.

Cancer Vaccines of the Invention

One embodiment of this invention is a method of treating cancer byadministering to a subject in need thereof an effective amount of animmune composition containing either Globo H or a fragment thereof(e.g., stage specific embryonic antigen-3 (SSEA-3, also known as Gb5),or SSEA-4) and an adjuvant. The types of target cancer include, but arenot limited to, breast cancer (including stages 1-4), lung cancer (e.g.,small cell lung cancer), liver cancer (e.g., hepatocellular carcinoma),oral cancer, stomach cancer (including T1-T4), colon cancer, nasopharynxcancer, skin cancer, kidney cancer, brain tumor (e.g., astrocytoma,glioblastoma multiforme, and meningioma), prostate cancer, ovariancancer, cervical cancer, bladder cancer, and endometrium,rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, and gastrointestinalstromal tumor.

Cancers classified by site include cancer of the oral cavity and pharynx(lip, tongue, salivary gland, floor of mouth, gum and other mouth,nasopharynx, tonsil, oropharynx, hypopharynx, other oral/pharynx);cancers of the digestive system (esophagus; stomach; small intestine;colon and rectum; anus, anal canal, and anorectum; liver; intrahepaticbile duct; gallbladder; other biliary; pancreas; retroperitoneum;peritoneum, omentum, and mesentery; other digestive); cancers of therespiratory system (nasal cavity, middle ear, and sinuses; larynx; lungand bronchus; pleura; trachea, mediastinum, and other respiratory);cancers of the mesothelioma; bones and joints; and soft tissue,including heart; skin cancers, including melanomas and othernon-epithelial skin cancers; Kaposi's sarcoma and breast cancer; cancerof the female genital system (cervix uteri; corpus uteri; uterus, nos;ovary; vagina; vulva; and other female genital); cancers of the malegenital system (prostate gland; testis; penis; and other male genital);cancers of the urinary system (urinary bladder; kidney and renal pelvis;ureter; and other urinary); cancers of the eye and orbit; cancers of thebrain and nervous system (brain; and other nervous system); cancers ofthe endocrine system (thyroid gland and other endocrine, includingthymus); lymphomas (Hodgkin's disease and non-Hodgkin's lymphoma),multiple myeloma, and leukemias (lymphocytic leukemia; myeloid leukemia;monocytic leukemia; and other leukemias).

Other cancers, classified by histological type, that may be suitabletargets for cancer vaccines according to the present invention include,but are not limited to, neoplasm, malignant; Carcinoma, NOS; Carcinoma,undifferentiated, NOS; Giant and spindle cell carcinoma; Small cellcarcinoma, NOS; Papillary carcinoma, NOS; Squamous cell carcinoma, NOS;Lymphoepithelial carcinoma; Basal cell carcinoma, NOS; Pilomatrixcarcinoma; Transitional cell carcinoma, NOS; Papillary transitional cellcarcinoma; Adenocarcinoma, NOS; Gastrinoma, malignant;Cholangiocarcinoma; Hepatocellular carcinoma, NOS; Combinedhepatocellular carcinoma and cholangiocarcinoma; Trabecularadenocarcinoma; Adenoid cystic carcinoma; Adenocarcinoma in adenomatouspolyp; Adenocarcinoma, familial polyposis coli; Solid carcinoma, NOS;Carcinoid tumor, malignant; Bronchiolo-alveolar adenocarcinoma;Papillary adenocarcinoma, NOS; Chromophobe carcinoma; Acidophilcarcinoma; Oxyphilic adenocarcinoma; Basophil carcinoma; Clear celladenocarcinoma, NOS; Granular cell carcinoma; Follicular adenocarcinoma,NOS; Papillary and follicular adenocarcinoma; Nonencapsulatingsclerosing carcinoma; Adrenal cortical carcinoma; Endometroid carcinoma;Skin appendage carcinoma; Apocrine adenocarcinoma; Sebaceousadenocarcinoma; Ceruminous adenocarcinoma; Mucoepidermoid carcinoma;Cystadenocarcinoma, NOS; Papillary cystadenocarcinoma, NOS; Papillaryserous cystadenocarcinoma; Mucinous cystadenocarcinoma, NOS; Mucinousadenocarcinoma; Signet ring cell carcinoma; Infiltrating duct carcinoma;Medullary carcinoma, NOS; Lobular carcinoma; Inflammatory carcinoma;Paget's disease, mammary; Acinar cell carcinoma; Adenosquamouscarcinoma; Adenocarcinoma w/ squamous metaplasia; Thymoma, malignant;Ovarian stromal tumor, malignant; Thecoma, malignant; Granulosa celltumor, malignant; Androblastoma, malignant; Sertoli cell carcinoma;Leydig cell tumor, malignant; Lipid cell tumor, malignant;Paraganglioma, malignant; Extra-mammary paraganglioma, malignant;Pheochromocytoma; Glomangiosarcoma; Malignant melanoma, NOS; Amelanoticmelanoma; Superficial spreading melanoma; Malig melanoma in giantpigmented nevus; Epithelioid cell melanoma; Blue nevus, malignant;Sarcoma, NOS; Fibrosarcoma, NOS; Fibrous histiocytoma, malignant;Myxosarcoma; Liposarcoma, NOS; Leiomyosarcoma, NOS; Rhabdomyosarcoma,NOS; Embryonal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromalsarcoma, NOS; Mixed tumor, malignant, NOS; Mullerian mixed tumor;Nephroblastoma; Hepatoblastoma; Carcinosarcoma, NOS; Mesenchymoma,malignant; Brenner tumor, malignant; Phyllodes tumor, malignant;Synovial sarcoma, NOS; Mesothelioma, malignant; Dysgerminoma; Embryonalcarcinoma, NOS; Teratoma, malignant, NOS; Struma ovarii, malignant;Choriocarcinoma; Mesonephroma, malignant; Hemangiosarcoma;Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma,malignant; Lymphangiosarcoma; Osteosarcoma, NOS; Juxtacorticalosteosarcoma; Chondrosarcoma, NOS; Chondroblastoma, malignant;Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma;Odontogenic tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma,malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma;Glioma, malignant; Ependymoma, NOS; Astrocytoma, NOS; Protoplasmicastrocytoma; Fibrillary astrocytoma; Astroblastoma; Glioblastoma, NOS;Oligodendroglioma, NOS; Oligodendroblastoma; Primitive neuroectodermal;Cerebellar sarcoma, NOS; Ganglioneuroblastoma; Neuroblastoma, NOS;Retinoblastoma, NOS; Olfactory neurogenic tumor; Meningioma, malignant;Neurofibrosarcoma; Neurilemmoma, malignant; Granular cell tumor,malignant; Malignant lymphoma, NOS; Hodgkin's disease, NOS; Hodgkin's;paragranuloma, NOS; Malignant lymphoma, small lymphocytic; Malignantlymphoma, large cell, diffuse; Malignant lymphoma, follicular, NOS;Mycosis fungoides; Other specified non-Hodgkin's lymphomas; Malignanthistiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferativesmall intestinal disease; Leukemia, NOS; Lymphoid leukemia, NOS; Plasmacell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myeloidleukemia, NOS; Basophilic leukemia; Eosinophilic leukemia; Monocyticleukemia, NOS; Mast cell leukemia; Megakaryoblastic leukemia; Myeloidsarcoma; and Hairy cell leukemia.

The term “treating” as used herein refers to the application oradministration of a composition including one or more active agents to asubject, who has cancer, a symptom of cancer, or a predisposition towardcancer, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve, or affect the cancer, the symptoms of thecancer, or the predisposition toward the cancer. “An effective amount”as used herein refers to the amount of each active agent required toconfer therapeutic effect on the subject, either alone or in combinationwith one or more other active agents. Effective amounts vary, asrecognized by those skilled in the art, depending on route ofadministration, excipient usage, and co-usage with other active agents.

The immune composition used in the above-described method can contain aglycan (i.e., a molecule containing a sugar moiety) that is Globo H or afragment thereof and an adjuvant. Globo H is a glycan containing thehexasaccharide epitope (Fucα1→2 Galβ→3 GalNAcβ1→3 Galα1→4 Galβ1→4 Glc),and optionally, a non-sugar moiety. Its fragment is a glycan containinga fragment of the hexasaccharide epitope and, if applicable, thenon-sugar moiety. These oligosaccharides can be prepared by routinemethods. (See Huang et al., Proc. Natl. Acad. Sci. USA 103:15-20(2006)). If desired, they can be linked to a non-sugar moiety.

The parent application U.S. patent application Ser. No. 12/485,546, wasbased on unexpected discoveries that (1) SSEA-3, the immediate precursorof Globo H, is expressed at a high level in breast cancer stem cells andtherefore can serve as a suitable target for breast cancer treatment,and (2) α-galactosyl-ceramide (α-GalCer) is an effective adjuvant thatpromotes production of anti-Globo H and anti-SSEA-3 antibodies.

U.S. patent application Ser. No. 12/485,546 features an immunecomposition containing Globo H or its fragment (e.g., SSEA-3) and anadjuvant (e.g., α-GalCer). Globo H or its fragment can be conjugatedwith Keyhole Limpet Hemocyanin (KLH). When administered into a subject(e.g., a human), this immune composition elicits immune responses (e.g.,antibody production) targeting Globo H or its fragment and, therefore,is effective in treating cancer (e.g., breast cancer, prostate cancer,ovarian cancer, and lung cancer).

U.S. patent application Ser. No. 12/485,546 relates to a method ofproducing antibody specific to Globo H or its fragment by administeringto a non-human mammal (e.g., mouse, rabbit, goat, sheep, or horse) theimmune composition described above and isolating from the mammalianantibody that binds to Globo H or its fragment.

The Globo H or other glycans described in the instant disclosure isconjugated to a protein carrier, such as DT-CRM197. They can then bemixed with an adjuvant, such as C34 and optionally a pharmaceuticallyacceptable carrier (e.g., a phosphate buffered saline, or a bicarbonatesolution) to form an immune composition (e.g., a vaccine) viaconventional methods. See, e.g., U.S. Pat. Nos. 4,601,903; 4,599,231;4,599,230; and 4,596,792. The composition may be prepared asinjectables, as liquid solutions, or emulsions and the carrier isselected on the basis of the mode and route of administration, as wellas on the basis of standard pharmaceutical practice. Suitablepharmaceutical carriers and diluents, and pharmaceutical necessities fortheir use, are described in Remington's Pharmaceutical Sciences. Theimmune composition preferably contains α-GalCer as an adjuvant. Otherexamples of adjuvant include, but are not limited to, a cholera toxin,Escherichia coli heat-labile enterotoxin (LT), liposome,immune-stimulating complex (ISCOM), or immunostimulatory sequencesoligodeoxynucleotides (ISS-ODN). The composition can also include apolymer that facilitates in vivo delivery. See Audran R. et al. Vaccine21:1250-5, 2003; and Denis-Mize et al. Cell Immunol., 225:12-20, 2003.When necessary, it can further contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, or pH buffering agentsto enhance the ability of the composition to elicit immune responsesagainst the sugar moiety in Globo H or its fragment. The immunecomposition described herein can be administered parenterally (e.g.,intravenous injection, subcutaneous injection or intramuscularinjection). Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkalene glycols ortriglycerides. Oral formulations may include normally employedincipients such as, for example, pharmaceutical grades of saccharine,cellulose, magnesium carbonate and the like. These compositions take theform of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and contain 10-95% of the immunecomposition described herein.

The immune composition described herein can be administered parenterally(e.g., intravenous injection, subcutaneous injection or intramuscularinjection). Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkalene glycols ortriglycerides. Oral formulations may include normally employedincipients such as, for example, pharmaceutical grades of saccharine,cellulose, magnesium carbonate and the like. These compositions take theform of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and contain 10-95% of the immunecomposition described herein.

The immune composition is administered in a manner compatible with thedosage formulation, and in an amount that is therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and if needed, toproduce a cell-mediated immune response. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner. However, suitable dosage ranges are readily determinableby one skilled in the art. Suitable regimes for initial administrationand booster doses are also variable, but may include an initialadministration followed by subsequent administrations. The dosage of thevaccine may also depend on the route of administration and variesaccording to the size of the host.

The immune composition of this invention can also be used to generateantibodies in animals for production of antibodies, which can be used inboth cancer treatment and diagnosis. Methods of making monoclonal andpolyclonal antibodies and fragments thereof in animals (e.g., mouse,rabbit, goat, sheep, or horse) are well known in the art. See, forexample, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York. The term “antibody” includes intactimmunoglobulin molecules as well as fragments thereof, such as Fab,F(ab)₂, Fv, scFv (single chain antibody), and dAb (domain antibody;Ward, et. al. (1989) Nature, 341, 544).

Globo H-DT-CRM197 and Related Vaccines

Globo H (1) and its fragments 2-10 were synthesized by methods describedherein. For protein conjugation, purified Globo H half ester 12 wasincubated with individual carrier proteins as shown in FIG. 14.

The Globo H-protein conjugates were characterized by MALDI-TOF analysisto determine the number of Globo H molecules on each carrier protein.The average number of Globo H incorporation is listed in Table 1.

TABLE 1 MALDI-TOF analysis of Globo H incorporation. Pro- After AverageCarbohy- tein Glyco- Incorpo- drate per- Ref MW sylation^(a) ration (n)centage GH-BSA 66431 66449 76029 8 14.4% GH-DT 58472 58326 62138 2~46.8% GH-TT 150682 155609 162902 6 4.5% GH-KLH*   8.6 × 10⁶ ~700 14.7%GH- 25 kD × 1600 N.D. Bamboo ^(a)Peak m/z in MALDI-TOF; N.D.: Notdetermined; *GH-KLH was provided by Optimer Inc.

The GH-KLH conjugate showed the greatest number of Globo Hincorporation, mostly due to the larger size and more Lys residues ofKLH. The same coupling procedure using p-nitrophenyl linker was alsoapplied to bamboo mosaic virus which contains more than 100,000 lysineresidues on the coat of virus. However, the instability of the viruswhile reacting in sodium phosphate buffer (pH=7.2) at 4° C. is a majorconcern for further development. Additionally, the GH-BaMV 16 limits itsdetection by MALDI-TOF analysis due to its tremendous size.

The synthetic Globo H and truncated fragments (FIG. 1) were attachedwith a pentylamine linker at the reducing ends and covalentlyimmobilized onto the NHS-coated glass slide. Nine of the elevenoligosaccharides were selected to be printed on the microarray. Eachmicroarray slide was spotted with 50 μM of nine Globo H analogs (SSEA-4,GH, Gb5, Gb4, Gb3, Gb2, BB4, BB3, and BB2) respectively in 12replications.

To validate the carbohydrates on the microarray, mouse monoclonalantibodies (VK9 and Mbr1 for Globo H, and anti-SSEA-3) were used andrespective secondary antibodies (goat anti-mouse IgG and IgM) were usedto examine the binding specificity, and the results are shown in FIG.2A-2C. The data suggested that VK9 and Mbr1 both recognized Globo H andthe outer tetrasaccharide BB4, though MBr1 also slightly recognized BB3.In addition, anti-SSEA-3 antibody specifically recognized SSEA-3 antigen(Gb5) without any cross reactivity. The results indicated that the GloboH microarray could be employed to profile the specificity and potency ofpolyclonal antibodies from immunized mice.

As previously reported, immunization of mice with a fully syntheticGlobo H vaccine and co-administered with QS-21 resulted in thegeneration of antibodies against human breast cancer cells; however themouse antibodies are mainly IgM, even after several boostingvaccinations. (Ragupathi G, et al. (1997) Angew Chem Int Ed 36:125-128).

A group of mice were immunized with 1 μg synthetic Globo H-conjugateswith or without the glycolipid adjuvant, α-GalCer (C1) subcutaneously.It was found that GH-KLH, GH-DT and GH-BV are the most effectiveimmunogens for IgM induction, followed by GH-TT, and GH-BSA assummarized in FIG. 3A, and α-GalCer is capable of stimulating the immuneresponse to induce high levels of IgM antibodies. A similar trend wasalso observed in mouse IgG antibodies (FIG. 3B), and the relative IgGlevels were higher than IgM levels. In brief, despite the lowercarbohydrate density of the synthetic glycoconjugate, GH-DT exhibited asimilar immunogenicity to GH-KLH, and the adjuvant α-GalCer was shown toenhance the immune response.

Since α-GalCer has been shown to be an effective adjuvant for GH-DT,other glycolipids with better adjuvant activity than C1 were examined asshown in FIG. 4. Groups of mice were immunized with GH-DT and GH-BV withor without glycolipids. Sera were obtained and introduced to glycanmicroarray analysis. In general, mouse anti-Globo H IgG titers increasedas immunization proceeded but the IgM levels were almost independent ofvaccination times (FIG. 5). Among the GH-BV vaccinated groups, there isno significant difference in the IgM level between glycolipid-vaccinetreatment and the vaccine alone. Although the results suggested thatGH-BV in combination with glycolipid was not an effective immunizationregimen, the poor immunogenicity may result from the unstable feature ofBaMV. Nevertheless, the α-GalCer analogs, especially 7DW8-5 cooperatedwell with GH-DT to induce mouse immune response.

Interestingly, the mouse polyclonal IgG antibodies generated by GH-DTand various glycolipid adjuvants not only neutralize Globo H but alsocross-react with Gb5, SSEA-4 and Gb4 and C34 appears to be the mosteffective glycolipid adjuvant (FIG. 6). In order to search for a newcomposition of vaccine that can induce much higher titer of IgG thanIgM, Globo H-DT conjugate and glycolipid C1 or C34 or commerciallyavailable adjuvant AlPO₄ (aluminium phosphate) or MF59 were tested.

Surprisingly, Globo H-DT with glycolipid C34 induces IgG antibody almostexclusively after the 3^(rd) vaccination (FIG. 7). To summarize, thenovel glycolipid adjuvant 7DW8-5 combined with GH-DT conjugates was ableto enhance both anti-Globo H IgG and IgM antibodies, and glycolipidadjuvant C34 combined with GH-DT can induce antibody titer of IgG muchhigher than IgM. They also exhibited diverse binding affinity to SSEA-3(Gb5) and SSEA-4 antigens, both specifically expressed on the surface ofbreast cancer stem cells.

In order to further compare the effect of different glycolipid adjuvantson Globo H vaccine, we immunized seven groups of mice with GH-KLH. Theresults suggested that mice vaccinated with glycolipids induced higherlevels of anti-Globo H antibodies (FIG. 8). Although MF59 is a strongadjuvant it failed to collaborate with GH-KLH to induce antibodiesagainst Globo H. AlPO₄ (aluminium phosphate) also showed no obviousimpact on the induction of antibodies. On the other hand, GH-KLH alongwith C34 showed superior immunogenicity after the first and secondvaccinations but exhibited no significant difference to C1 after thethird vaccination. Overall, these findings suggest the potential ofnovel glycolipid derivatives as adjuvants for carbohydrate basedvaccines.

The nature of cellular and humoral immune response is influenced notonly by antigen and adjuvant combinations but also by the carrier androute of immunization. As Sesardic and co-workers described, DT-CRM197,a mutant toxin devoid of toxic activity induces antigen-specific T cellproliferation and elevates splenocyte production of IL-2, IFN-γ andIL-6, suggesting its role in Th1 driven pathway. (Miyaji E N et al.(2001) Infect Immun 69:869-874; Godefroy S, et al. (2005) Infect Immun73:4803-4809; Stickings P, et al. (2008) Infect Immun 76:1766-1773.)Despite the fact that the cytokine profile was predominantly Th1,subclasses of anti-CRM197 antibodies were IgG1 with no detectable IgG2a,which suggests a mixed Th1/Th2 response. These results prompted theevaluation of the antibody isotype profile of the Globo H vaccines, andpresent studies showed that GH-DT or GH-KLH in combination withglycolipid adjuvants induced mainly IgG1 antibody with a trace amount ofIgG2a (FIG. 9).

Despite the fact that glycolipid adjuvants enhanced Th1 biased cytokinessecretion when administrated alone intravenously (i.v.), the antibodyclass switch (IgG2a) was not observed. Overall, the glycolipids play apivotal role in enhancing both cellular and humoral immune response.

Globo H, SSEA-3 and SSEA-4 Cancer Vaccines

SSEA-3 (Gb5) and SSEA-4 conjugated with DT were synthesized and tested.After 3^(rd) vaccination, antibodies titer of IgM and IgG were comparedand it was found that SSEA-3-DT and SSEA-4-DT also induced much highertiter of IgG than IgM (FIG. 10).

Since GH-DT and C34 induced antibodies to recognize Globo H, Gb5 andSSEA-4, the specificity of SSEA-3-DT and SSEA-4-DT vaccines in thepresence of adjuvants using an array of 24 glycans were examined withfocus on the study of IgG (FIG. 11).

As shown in FIG. 12, mice immunized with Globo H-DT and C34 adjuvantinduced antibodies that can recognize Globo H, SSEA-3 (Gb5) and SSEA-4with high selectivity, and vaccine SSEA-3-DT with adjuvant MF59 inducedhigh immune response with low selectivity. On the other hand, SSEA-3-DTcombined with adjuvant C34 only induced antibodies against Globo H,SSEA-3, and SSEA-4.

Interestingly, SSEA-4-DT (sialyl-Gb5) in the presence or absence ofadjuvants induced IgG and IgM antibodies specifically recognizing SSEA-4and its truncated structures (SSEA-4 with head lactose deletion).Without being bound by theory, it is postulated that sialic acid ishighly immunogenic and induces highly specific immune response.

Immunization of mice with SSEA-3-DT-C34 induced antibodies reactive withGlobo H, SSEA-3 and SSEA-4, suggesting that a Globo H-based vaccine cantarget tumor cells and breast cancer stem cells expressing Globo H,SSEA-3 and SSEA-4.

Immunization of mice with Globo H-DT-C34 induced antibodies reactivewith Globo H, SSEA-3 and SSEA-4, suggesting that a Globo H-based vaccinecan target tumor cells and breast cancer stem cells expressing Globo H,SSEA-3 and SSEA-4.

Immunization of mice with SSEA-4-DT induced antibodies reactive withSSEA-4, suggesting that a SSEA-4-DT-based vaccine can target tumor cellsand breast cancer stem cells expressing SSEA-4.

Tumor Size Reduction by Cancer Vaccines

In order to directly assess the efficacy of the synthetic glycoconjugatevaccines, the tumor sizes were measured three times per week as shown inFIG. 13. In general, tumor grows 2 weeks after injection with 4T1, aGlobo H bearing breast cancer cell line. All the vaccinated groups alongwith glycolipid adjuvants still showed comparative smaller tumorprogression compared to GH-DT alone and PBS control at day 24. The datasuggest that vaccination with GH-DT and a glycolipid adjuvant delayedsome degree of the tumor progression in vivo.

Expression of SSEA-3 and SSEA-4 in Breast Cancer and BCSCs

The expression of Globo H in BCSCs, but at a lower frequency thannon-BCSCs, and a higher frequency of SSEA-3 expression than Globo Hexpression in breast cancer and BCSCs has been shown. (Chang W-W. etal., (2008) Proc Natl Acad Sci USA 105(33):11667-11672, incorporatedherein by reference in its entirety.)

The clinical characteristics of 35 patients with breast cancer in whomrange of SSEA-3 or SSEA-4 expression was measured, are summarized inTable 2. The median age was 48 years (ranging from 31 to 82 years). Theyconsisted of 1 stage 0, 10 stage I, 19 stage II, and 5 stage III. Amajority of the tumor specimens had the pathology of infiltrating ductalcarcinoma (80.0%), with 51.4% positive for ER and 65.7% positive fornode involvement. In Table 2, the ranges of SSEA-3 or SSEA-4 expressionis represented by the percentage of positive cells within total cancercells. A t test was used for statistical analysis of SSEA-3 or SSEA-4expression relative to HER-2 or nodal involvement status. HER-2expression was determined by immunohistochemistry. There was nosignificant correlation between expression level of SSEA-3 or SSEA-4 ontumors and various clinico-pathological factors, such as stage (SSEA-4:P=0.3498; SSEA-3, P=0.9311), or HER-2 (SSEA-4: P=0.0142; SSEA-3,P=0.0128) (Table 2).

TABLE 2 Clinical characteristics of patients with breast cancer. SSEA4SSEA3 Percent cells Percent cells with expression with expressionCharacteristic No. % Median (range) P value Median (range) P valuePatients enrolled 35 100 Age, years Median 48 Range 31-82 Tumor typeInfiltrating ductal carcinoma 28 80.0 Infiltrating lobular carcinoma 12.8 Ductal carcinoma in situ 1 2.8 Medullary carcinoma 1 2.8 Atypicalmedullary carcinoma 1 2.8 Metaplastic carcinoma 2 6.0 Inflammatorycarcinoma 1 2.8 Stage 0.7880 0.9311 0 1 2.9 33.1 (33.1)     1.4(1.4)     I 10 28.6 41.4 (0.5-69.1) 36.4 (0.0-55.9) II 19 54.3 39.3(0.0-77.1) 30.9 (0.0-66.4) III 5 14.2 49.8 (7.7-70.7) 32.3 (0.0-36.1)Node involvement 0.0322 0.4925 Negative 23 65.7 37.8 (0.0-69.1) 30.9(0.0-66.4) Positive 12 34.3  49.1 (17.4-77.1) 35.8 (0.0-60.7) ER 0.01420.0128 Negative 18 51.4 36.2 (0.5-60.3) 29.7 (0.0-38.6) Positive 17 48.648.5 (0.0-77.1) 40.0 (0.0-66.4)

Primary tumor cells isolated from enrolled patients by enzymaticdigestion were stained with specific antibodies to CD45, CD24, CD44, andCD45′ cells were first gated out to eliminate the leukocytes. To comparethe SSEA-3 or SSEA-4 expression between BCSCs and non-BCSCs, CD45 tumorcells were further separated into BCSCs and non-BCSCs based on theirexpressions of surface markers. The BCSCs were identified asCD45⁻/CD24⁻/CD44⁺ cells; the rest of the CD45 population were consideredas non-BCSCs.

Using this approach, the expression of SSEA-3 or SSEA-4 in BCSCs andnon-BCSCs were evaluated in 35 tumor specimens. Overall, SSEA-4 wasdetected in 34/35 (97.1%) and SSEA-3 in 27/35 (77.1%) of the tumors(Table 3). SSEA4 or SSEA3 expression was determined by flow cytometry.BCSCs were defined as CD45⁻CD24⁻CD44⁺ cells and non-BCSCs were definedas the remaining populations of CD45⁻ cells. Range was calculated aspercentage of positive cells in total cells.

As summarized in Table 3, among the 27/35 (77.1%) samples expressingSSEA-3, the percentage of positive cells ranged from 1.4% to 66.4%. Thenon-BCSCs isolated from 25/35 tumors expressed SSEA-3, with thepercentage of positive cells ranging from 24.3% to 70.4%. In comparison,BCSCs from 23 of 35 (65.7%) tumors showed positive staining for SSEA-3,with the percentage of positive cells ranging from 5.0% to 58.4%.

Among the 34/35 (97.1%) samples expressing SSEA-4, the percentage ofpositive cells ranged from 0.5% to 77.1%. The non-BCSCs isolated from32/35 tumors expressed SSEA-4, with the percentage of positive cellsranging from 24.0% to 78.1%. In comparison, BCSCs from 31 of 35 (88.6%)tumors showed positive staining for SSEA-4, with the percentage ofpositive cells ranging from 5.6% to 83.6%.

TABLE 3 Comparison of SSEA4 and SSEA3 expression in BCSCs and non-BCSCsPositive Glycan percent Cells with % of Population No. of patients No.expression median (Range) Total SSEA-4 Total 35 34 41.4 (0.5-77.1) 97.1Non-BCSCs 35 32 43.7 (4.0-78.1) 91.4 BCSCs 35 31 37.1 (5.6-83.6) 88.6SSEA-3 Total 35 27 36.4 (1.4-66.4) 77.1 Non-BCSCs 35 25  40.5(24.3-70.4) 71.4 BCSCs 35 23 24.3 (5.0-58.4) 65.7Expression of SSEA-4 in BCSCs

To compare the SSEA-4 expression between BCSCs and non-BCSCs, CD45 tumorcells were further separated into BCSCs and non-BCSCs based on theirexpressions of surface markers. The BCSCs were identified asCD45⁻/CD24⁻/CD44⁺ cells; the rest of the CD45 population were consideredas non-BCSCs. The expression of SSEA-4 within each of these two gatedpopulations varied among tumor samples a shown in FIG. 15. For instance,BCSCs of patient BC0264, which accounted for 5.7% of the total isolatedtumor cells, were negative for SSEA-4, whereas 60.3% of the non-BCSCsexpressed SSEA-4. For patient BC0266, SSEA-4 expression was detected in59.4% of non-BCSCs and 55.7% of BCSCs. For patient BC0313, SSEA-4expression was detected in 32.4% of non-BCSCs and 83.6% of BCSCs.Altogether, SSEA-4 was detected in 34/35 (97.1%) samples tested with thepercentage of positive cells ranging from 0.5% to 77.1%). (Table 32).

Expression of SSEA-3 and SSEA-4 in Normal Tissues

Using tissue microarray, SSEA-4 expression was analyzed among 20different organs by immunohistochemical staining, as shown in Table 4(E, epithelial; C, connective tissue).

TABLE 4 Expression of SSEA-4 in normal tissues Antigen Normal tissueSSEA4 Brain 0/5 Bone 0/5 Lymph node 0/5 E C Breast 1/5 0/5 Colon* 2/40/4 Esophagus 0/5 0/5 Intestine 5/5 0/5 Kidney 2/5 0/5 Liver 0/5 0/5Lung 1/5 0/5 Ovary 1/5 0/5 Pancreas 1/5 0/5 Prostate 0/5 0/5 Rectum 5/50/5 Skin 0/5 0/5 Spleen 0/5 0/5 Stomach 4/5 0/5 Testis 4/5 0/5 Thymusgland 1/5 0/5 Uterine cervix 1/5 0/5

SSEA-4 is expressed on the epithelial cells of several glandulartissues, such as breast, colon, gastrointestinal tract, kidney, lung,ovary, pancreas, rectum, stomach, testes, thymus and uterine cervix(Table 4). Further, in a manner similar to Globo H and SSEA-3 (ChangW-W. et al., (2008) Proc Natl Acad Sci USA 105(33):11667-11672), SSEA-4expression was confined mainly to the cytoplasm or apical surface ofepithelial cells which were essentially inaccessible to the immunesystem, as shown in FIG. 16.

By comparison, Globo H is expressed on the epithelial cells of severalglandular tissues, such as breast, gastrointestinal tract, pancreas,prostate, and uterine cervix. The distribution of SSEA3 is similar tothat of Globo H except for its absence in normal breast tissues butpresence in kidney, rectum, testis, and thymus, which were negative forGlobo H (Chang W-W. et al., (2008) Proc Natl Acad Sci USA105(33):11667-11672).

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

General Methods, Materials and Instrumentation

Materials

Commercial solvents and reagents were used as received without furtherpurification and purchased from Sigma-Aldrich, Acros, Merck, Echochemical and Senn Chemical. Monoclonal antibody Mbr1 was purchased fromALEXIS biochemicals, Cy3-conjugated anti-mouse IgG (IgG, IgG1, andIgG2a) and IgM antibodies were from Jackson Immuno Research. DT-CRM197Protein and Tetanus toxoid were purchased from Merck and Adimmune,respectively. Aluminium phosphate gel adjuvant (AlPO₄) was purchasedfrom Brenntag Biosector. Bamboo virus and VK9 monoclonal antibody wereprepared from Dr. Lin's and Dr. Yu's laboratory, respectively.Glycolipid derivatives were synthesized and provided by Dr. Wong'slaboratory.

General Methods

Molecular sieves (MS, AW-300) used in glycosylations were crushed andactivated before use. Reactions were monitored with analytical TLCplates (PLC silica gel-60, F₂₅₄, 2 mm, Merck) and visualized under UV(254 nm) or by staining with p-anisaldehyde. Flash column chromatographywas performed on silica gel (40-63 μm) or LiChroprep RP18 (40-63 μm).Dialysis membrane (Cellulose Ester, MCCO=10,000) was washed by ddH₂Obefore use.

Instrumentation

Proton nuclear magnetic resonance (¹H NMR) spectra, carbon nuclearmagnetic resonance (¹³C NMR) spectra were recorded by Bruker Advance 600(600 MHz/150 MHz) NMR spectrometers. Chemical shifts for protons arereported in ppm (δ scale) and referenced to tetramethylsilane (δ=0).Chemical shifts for carbon are also reported in parts per million (ppm,6 scale). DEPT 135 (Distortion-less enhancement by polarizationtransfer) was employed for determination of multiplicity. Data arerepresented as follows: chemical shift, multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, m=multiplet, br=broad), integration andcoupling constant (J) in Hz. High resolution mass spectra were obtainedby BioTOF III, and the MALDI-TOF MS were employed by Ultraflex IITOF/TOF200.

Example 1 Synthesis of Globo H Conjugated with Different CarrierProteins

Globo H (1; see FIG. 11) and its fragments 2-10 were synthesized byusing a programmable one-pot strategy. (Huang C-Y, et al. (2006) ProcNatl Acad Sci USA 103:15-20.) The reaction of 1 was carried with anefficient homobifunctional linker in anhydrous DMF solution at roomtemperature. (Wu X, et al. (2004) Org Lett 6:4407-4410; Wu X, Bundle D R(2005) J Org Chem 70:7381-7388.) The reaction was readily monitored byTLC. Once the disappearance of the free amine with a larger R_(f)product occurred, the reaction mixture was evaporated to remove DMF, andwashed with dichloromethane and water to remove the excess amount oflinker. Finally, the product was purified by reverse phase (C18) columnchromatography, and gradually eluted with water containing 1% aceticacid to 40% methanol in water. The solution was then lyphophilized toyield the light yellow product 12. Finally, for protein conjugation, thepurified Globo H half ester 12 (30-40 equiv) was incubated withindividual carrier proteins in phosphate buffer (10 mM, pH 7.2) for 24hours at room temperature (FIG. 14). Importantly, the proteinconcentration must be adjusted to ˜5 mg/mL to maximize the coupling oflysine residues with Globo H half ester. After 24 h, the glycoconjugateswere then diluted, and dialyzed against deionized water to remove theremaining of p-nitrophenyl group. The solution was then lyphophilized toa white powder to give 13, 14, and 15.

The Globo H-protein conjugates were characterized by MALDI-TOF analysisto determine the number of Globo H molecules on each carrier protein.The average number of Globo H incorporation is listed in Table 1 shownsupra.

The glycoconjugates 13, 14, 15 were dissolved in ddH₂O to yield a finalconcentration around 1 μmol/μL. Sinapinic acid was selected as a matrixand mixed with freshly prepared acetonitrile and deionized water (1:1v/v) to make the final matrix concentration in 10 mg/mL including 0.1%TFA. Each sample was detected under a linear positive mode to get them/z spectrum. The molecular weight of each glycoconjugate was determinedby m/z. The glycoconjugate 14 showed heterogeneity, indicating anaverage of 2-4 incorporations. The GH-KLH conjugate showed the greatestnumber of Globo H incorporation, mostly due to the larger size and moreLys residues of KLH. The same coupling procedure using p-nitrophenyllinker was also applied to bamboo mosaic virus which contains more than100,000 lysine residues on the coat of virus. However, the instabilityof the virus while reacting in sodium phosphate buffer (pH=7.2) at 4° C.is a major concern for further development. Additionally, the GH-BaMV 16limits its detection by MALDI-TOF analysis due to its tremendous size.Finally, the lyphophilized glycoconjugates were stored at −30° C. andreconstituted with sterile water before immunization.

Example 2 Glycan Microarray Fabrication and Validation

The synthetic Globo H and truncated fragments (FIG. 1) were attachedwith a pentylamine linker at the reducing ends and covalentlyimmobilized onto the NHS-coated glass slide. Nine of the elevenoligosaccharides were selected to be printed on the microarray. Serialoligosaccharide concentrations (1, 5, 10, 20, 40, 50, 80, 100 μM) weretested to optimize the binding affinity and fluorescence intensity. Eachmicroarray slide was spotted with 50 μM of nine Globo H analogs (SSEA-4,GH, Gb5, Gb4, Gb3, Gb2, BB4, BB3, and BB2) respectively in 12replications. After reaction in 80% humidity atmosphere, the slides werestored at room temperature in desiccators before use.

To validate the carbohydrates on the microarray, mouse monoclonalantibodies (VK9 and Mbr1 for Globo H, and anti-SSEA-3) and respectivesecondary antibodies (goat anti-mouse IgG and IgM) were used to examinethe binding specificity, and the results are shown in FIGS. 2A-2C. Thedata suggests that VK9 and Mbr1 both recognized Globo H and the outertetrasaccharide BB4, although MBr1 also slightly recognized BB3.(Gilewski T el al. (2001) Proc Natl Acad Sci USA 98:3270-3275; HuangC-Y, et al. (2006) Proc Natl Acad Sci USA 103:15-20.) In addition,anti-SSEA-3 antibody specifically recognized SSEA-3 antigen (Gb5)without any cross reactivity. The results indicated that the Globo Hmicroarray could be employed to profile the specificity and potency ofpolyclonal antibodies from immunized mice.

Example 3 Mouse Immunization

In this study, a group of mice was immunized with 1 μg synthetic Globo H(GH)-conjugates with or without the glycolipid adjuvant, α-GalCer (C1)subcutaneously. Ten days after three vaccinations at weekly intervals,mice sera were collected and subsequently introduced to the glycanmicroarray to evaluate the antibody levels. It was found that GH-KLH,GH-DT and GH-BV are the most effective immunogens for IgM induction,followed by GH-TT, and GH-BSA as summarized in FIG. 3A, and α-GalCer iscapable of stimulating the immune response to induce high levels of IgMantibodies. A similar trend was also observed in mouse IgG antibodies(FIG. 3B), and the relative IgG levels were higher than IgM levels. Inbrief, despite the lower carbohydrate density of the syntheticglycoconjugate, GH-DT exhibited a similar immunogenicity to GH-KLH, andthe adjuvant α-GalCer was shown to enhance the immune response.

Since C1 was shown to be an effective adjuvant for GH-DT, otherglycolipids with better adjuvant activity than C1 were examined as shownin FIG. 4. (Fujio M, et al. (2006) J Am Chem Soc 128:9022-9023.)

Groups of mice were immunized intramuscularly with 1.6 μg of GH-DT andGH-BV with or without 2 μg of glycolipids twice a week. Sera wereobtained two weeks after the third vaccination and introduced to glycanmicroarray analysis. In general, mouse anti-Globo H IgG titers increasedas immunization proceeded but the IgM levels were almost independent ofvaccination times (FIG. 5). Among the GH-BV vaccinated groups, there wasno significant difference in the IgM level between glycolipid-vaccinetreatment and the vaccine alone. Although the results suggested thatGH-BV in combination with glycolipid was not an effective immunizationregimen, the poor immunogenicity may result from the unstable feature ofBaMV. Nevertheless, the α-GalCer analogs, especially 7DW8-5 cooperatedwell with GH-DT to induce mouse immune response.

Interestingly, the mouse polyclonal IgG antibodies generated by GH-DTand various glycolipid adjuvants not only neutralize Globo H but alsocross-react with Gb5, SSEA-4 and Gb4 and C34 appears to be the mosteffective (FIG. 6). In order to search for a new composition of vaccinethat can induce much higher titer of IgG than IgM, Globo H-DT conjugateand glycolipid C1 or C34 and commercially available adjuvant AlPO₄(aluminium phosphate) or MF59 were tested. Surprisingly, Globo H-DT withglycolipid C34 can induce almost IgG antibody after 3^(rd) vaccination(FIG. 7). In summary, the novel glycolipid adjuvant 7DW8-5 combined withGH-DT conjugates was able to enhance both anti-Globo H IgG and IgMantibodies, and glycolipid adjuvant C34 combined with GH-DT can induceantibody titer of IgG much higher than IgM. They also exhibited diversebinding affinity to Gb5 and SSEA-4 antigens, both specifically expressedon the surface of breast cancer stem cells.

In order to further compare the effect of different glycolipid adjuvantson Globo H vaccine, seven groups of mice with GH-KLH. were immunized Theresults suggested that mice vaccinated with glycolipids induced higherlevels of anti-Globo H antibodies (FIG. 8). Although MF59 is a strongadjuvant it failed to collaborate with GH-KLH to induce antibodiesagainst Globo H. AlPO₄ (aluminium phosphate) also showed no obviousimpact on the induction of antibodies. On the other hand, GH-KLH alongwith C34 showed superior immunogenicity after the first and secondvaccinations but exhibited no significant difference to C1 after thethird vaccination.

DT-CRM197, a mutant toxin devoid of toxic activity inducesantigen-specific T cell proliferation and elevates splenocyte productionof IL-2, IFN-γ and IL-6, suggesting its role in Th1 driven pathway.(Miyaji E N et al. (2001) Infect Immun 69:869-874; Godefroy S, et al.(2005) Infect Immun 73:4803-4809; Stickings P, et al. (2008) InfectImmun 76:1766-1773.) Despite the fact that the cytokine profile waspredominantly Th1, subclasses of anti-CRM197 antibodies were IgG1 withno detectable IgG2a, which suggests a mixed Th1/Th2 response. Theseresults prompted the evaluation of the antibody isotype profile of theGlobo H vaccines, and present studies showed that GH-DT or GH-KLH incombination with glycolipid adjuvants induced mainly IgG1 antibody witha trace amount of IgG2a (FIG. 9).

Despite the fact that glycolipid adjuvants enhanced Th1 biased cytokinessecretion when administrated alone intravenously (i.v.), the antibodyclass switch (IgG2a) was not observed. Overall, the glycolipids play apivotal role in enhancing both cellular and humoral immune response.

Gb5 and SSEA-4 conjugated with DT were also synthesized by the samestrategy. After 3^(rd) vaccination, antibodies titer of IgM and IgG werecompared and it was found that Gb5-DT and SSEA-4-DT also induced muchhigher titer of IgG than IgM (FIG. 10).

Example 4 Specificity Studies of Antibodies Induced by Different VaccineComposition

Since GH-DT and C34 induced antibodies to recognize Globo H, Gb5(SSEA-3) and SSEA-4, the specificity of SSEA-3-DT and SSEA-4-DT vaccinesin the presence of adjuvants using an array of 24 glycans with focus onthe study of IgG were next examined (FIG. 11).

As shown in FIG. 12, mice immunized with Globo H-DT and C34 adjuvantinduced antibodies that can recognize Globo H, SSEA-3 (Gb5) and SSEA-4with high selectivity, and vaccine SSEA-3-DT with adjuvant MF59 inducedhigh immune response with low selectivity. On the other hand, SSEA-3-DTcombined with adjuvant C34 only induced antibodies against Globo H,SSEA-3, and SSEA-4.

Interestingly, SSEA-4-DT in the presence or absence of adjuvants inducedIgG and IgM antibodies specifically recognizing SSEA-4 and its truncatedstructures (SSEA-4 with head lactose deletion). It is however not clearabout the origin of the selectivity.

In order to directly assess the efficacy of the synthetic glycoconjugatevaccines, the tumor sizes three times per week were measured as shown inFIG. 13. In general, tumor grows 2 weeks after injection with 4T1, aGlobo H bearing breast cancer cell line. All the vaccinated groups alongwith glycolipid adjuvants still showed comparative smaller tumorprogression compared to GH-DT alone and PBS control at day 24. Thepreliminary data suggested that vaccination with GH-DT and a glycolipidadjuvant indeed delayed some degree of the tumor progression in vivo.

Example 5 Preparation of Globo H Half Ester

The GloboH half ester was prepared as follows:

Globo H amine 1 (5 mg, 4.54 μmol) was dissolved in anhydrous DMFsolution. p-nitrophenyl ester linker (8.8 mg, 22.7 μmol) was then addedand stirred for 1-3 hours at room temperature. The reaction wasmonitored by TLC (1% AcOH in methanol) and Ninhydrin test. Thedisappearance of free amine with a larger R_(f) product indicated thecompletion of the reaction. The reaction mixture was evaporated underreduced pressure without heating to remove DMF, and then extracted withCH₂Cl₂ and water containing 1% of acetic acid twice. The water solutionwas concentrated and purified by reverse phase (C18) columnchromatography, and gradually eluted with H₂O containing 1% of aceticacid to MeOH:H₂O=4:6. The solution was then lyphophilized to a lightyellow solid product 12 (5.4 mg, Yield 88%)¹H NMR (600 MHz, D₂O) δ 8.25(d, 2H, J=9.0 Hz), 7.28 (d, 2H, J=9.0 Hz), 5.12 (d, 1H, J=3.9 Hz), 4.79(d, 1H, J=3.7 Hz), 4.51 (d, 1H, J=7.7 Hz), 4.44 (d, 1H, J=7.7 Hz), 4.39(d, 1H, J=7.7 Hz), 4.31-4.28 (t, 2H, J=7.7 Hz), 4.15-4.11 (m, 2H), 3.99(d, 1H, J=2.0 Hz), 3.92 (d, 1H, J=2.8 Hz), 3.89-3.44 (m, 33H), 3.16 (t,1H, J=8.6 Hz), 3.10 (t, 2H, J=6.7 Hz), 2.62 (t, 2H, J=6.9 Hz), 2.20 (t,2H, J=6.6 Hz), 1.93 (s, 3H), 1.62-1.49 (m, 4H) 1.54-1.48 (m, 2H),1.45-1.40 (m, 2H), 1.30-1.24 (m, 2H), 1.11 (d, 3H, J=6.5 Hz)¹³C NMR (150MHz, D₂O) δ178.0, 176.1, 176.0, 156.9, 147.1, 127.3, 124.5, 105.7,105.0, 103.7, 103.6, 102.2, 101.0, 80.5, 80.0, 78.9, 78.0, 77.8, 77.1,76.7, 76.4, 76.3, 76.2, 75.2, 74.6, 73.8, 73.5, 72.5, 72.1, 71.8, 71.2,70.9, 70.8, 70.1, 69.7, 69.5, 68.5, 62.6, 62.6, 62.0, 62.0, 61.7, 53.3,40.8, 37.1, 35.0, 30.0, 29.7, 26.4, 25.0, 24.1, 23.9, 17.0 HRMS:C₅₅H₈₇N₃O₃₅Na [M+Na]⁺ calculated: 1372.5018. found: 1372.5016.

Example 6 General Procedure for Generating Glycoconjugates

Glycoconjugates were manufactured as follows:

BSA, DT-CRM197, and Tetanus toxoid (Adimmune, Taiwan) was dissolved in100 mM phosphate buffer pH 7.2 (˜5 mg/ml), and 30 to 40 equivalents ofGlobo H half ester 35 were added to the solution. The mixture wasstirred gently for 24 h at room temperature. The mixture was thendiluted with deionized water and dialyzed against 5 changes of deionizedwater. The solution was then lyphophilized to a white powder. Theobtained Globo H-protein conjugates can be characterized by MALDI-TOFanalysis to determine the carbohydrate incorporation rate. 41 (GH-BSA),MALDI-TOF found 76029, 42 (GH-DT-CRM197) found 62138, 43 (GH-TT) found162902, 44(GH-BaMV) was not determined

Example 7 MALDI-TOF MS Analysis for Glycoconjugates

The glycoconjugates 41, 42, 43 and primary carrier proteins werereconstituted with ddH₂O (˜1 μg/μl). The matrix, sinapinic acid, wasfreshly prepared with acetonitrile and deionized water 1:1, making finalmatrix concentration in 10 mg/ml including 0.1% TFA. Gently loaded andmixed the matrix solution and glycoconjugates, then air dried the plate.Calibration was imperative using bovine serum albumin beforemeasurement. Each glycoconjugate and primary protein sample was detectedunder linear positive mode. The average molecular weight allows thecalculation of the average number of carbohydrate incorporated on thecarrier protein.

Example 8 Glycan Microarray Fabrication

Microarray were printed (BioDot, Cartesian Technologies, USA) by roboticpin (SMP3, TeleChem International Inc., USA) deposition of ˜0.7 nL ofvarious concentrations of amine-containing glycans in printing buffer(300 mM phosphate buffer, pH 8.5 containing 0.005% Tween-20) from a 96well onto NHS-coated glass slides. Each microarray slide was spottedwith 50 μM of nine Globo H analogs (SSEA-4, GH, Gb5, Gb4, Gb3, Gb2, BB4,BB3, and BB2) respectively in 12 replications. Printed slides wereallowed to react in an atmosphere of 80% humidity for an hour followedby desiccation overnight. These slides were stored at room temperaturein a dessicator before used.

Example 9 Serologic Assay (Glycan Microarray)

Mice sera were diluted 1:60 with 0.05% Tween 20 in 3% BSA/PBS buffer (pH7.4) as preliminary screening. The glycan microarray was blocked with 50mM ethanolamine for 1 h, and washed twice with ddH₂O and PBS bufferbefore used. The serum dilutions were then introduced to the Globo Hmicroarray, and incubated at room temperature for 1 h. The microarrayslides were further washed three times with PBST (0.05% Tween-20 in PBSbuffer) and PBS buffer, respectively. Next, Cy3-affiniPure goatanti-mouse IgG (H+L), IgG1, IgG2a or anti-mouse IgM was added to themicroarray slide and then sealed for 1 hour incubation at roomtemperature. Finally, the slides were washed three times with PBST, PBSand ddH₂O in sequence. The microarray slides were dried before scannedat 532 nm with a microarray fluorescence chip reader (Genepix 4000B).Data were analyzed by software GenePix Pro 6.0 (Axon Instruments, UnionCity, Calif., USA). To acquire the accurate measurement, PMT gain wasadjusted to 400 avoiding fluorescence saturation. The local backgroundwas subtracted from the signal at each glycan spot. The spots withobvious defects or no detectable signal were omitted. The ultimatefluorescence intensity was defined as the average of “medians of F532nm-B532 nm” from replicate spots.

Example 10 Serologic Assay (Enzyme-Linked Immunosorbent Assay)

0.2 μg of Globo-H ceramide in 100 μl carbonate bicarbonate buffer (pH10) was coated in 96-well plate (NUNC) at 4° C. for overnight. Washedwith PBS and blocked with 3% bovine serum albumin for 30 minutes at roomtemperature. Serial dilutions of mice sera were added into each well andincubated for 1 h at room temperature, followed by washing with DPBST(Dulbecco's Phosphate Buffered Saline, 0.05% Tween20). Goat anti-mouseIgG-AP (1:200, Southern Biotech., USA) was added and incubated for 45minutes at room temperature. The plates were washed with PBST five timesand then incubated with alkaline phosphatase substrate, p-nitrophenylphosphate (Sigma) for 8 minutes at 37° C. After incubation, the reactionwas stopped by adding 3 M NaOH solution and the plates were read at 405nm on the ELISA reader (SpectraMax, Molecular Devices) The titer wasdefined as the highest dilution yielding an optical density greater than0.1.

Example 11 Dosage and Immunization

(1) Groups of three mice (6-week-old female C57BL/6 mice, BioLASCO,Taiwan) were administered subcutaneously to abdomen region with GH-KLH(Optimer Inc.), GH-BSA, GH-TT, GH-CRM197, and GH-BaMV respectively withor without glycolipid adjuvant C1 or 7DW8-5 for three times with weeklyinterval. Each vaccination contained 1 μg of Globo H and with or without2 μg glycolipid adjuvant. Control mice were injected with phosphatebuffer saline (PBS) only. Mice were bled before first immunization(pre-immune) and ten days after third immunization. (2) Groups of threemice (8-week-old female Balb/c mice, BioLASCO, Taiwan) were immunizedintramuscularly three times at two weeks interval with GH-BaMV orGH-CRM197 with or without C1, C23, or 7DW8-5, respectively. Eachvaccination contained 1.6 μg of Globo H and with or without 2 μg ofadjuvant. Control mice were injected with phosphate buffer saline (PBS).Mice were bled before immunization and 2 weeks after each vaccination.(3) Groups of three mice (8-week-old female Balb/c mice, BioLASCO,Taiwan) were immunized with GH-CRM197 or GH-KLH with or without adjuvantC1, C17, 7DW8-5, C30, AlPO₄, MF59 (1:1 mixture) as (2) described. Allthe sera were obtained by centrifugation under 4000 g for 10 minutes.The serologic responses were analyzed by glycan microarray or comparedwith conventional ELISA assay.

Example 12 Xenograft Model

(1) Five groups of immunized female Balb/c mice (PBS, GH-CRM197 alone orwith C1, C23 and 7DW8-5, respectively) were injected with 2×10⁵metastatic mouse mammary tumor cell lines, 4T1 (in sterile PBS)subcutaneously 8 weeks after final vaccination. (2) Seven groups ofimmunized female Balb/c mice (GH-KLH alone or with C1, C17, 8-5, C30,AlPO₄ and MF59, respectively) were injected with 2×10⁵ metastatic mousemammary tumor cell lines, 4T1 (in sterile PBS) subcutaneously 6 weeksafter final vaccination. Mice anti-Globo H sera were monitored beforeand after tumor xenografting. Mice tumor size was measured by Verniercaliper three times per week and defined as (length×height×width)/2(mm³).

Example 13 Isolation of Primary Tumor Cells from Human Breast CancerSpecimens

Human breast cancer specimens were obtained from patients who hadundergone initial surgery at the Tri-Service General Hospital (Taipei,Taiwan). Samples were fully encoded to protect patient confidentialityand were used under a protocol approved by the Institutional ReviewBoard of Human Subjects Research Ethics Committee of Academia Sinica,Taipei, Taiwan. The tumor specimens were sliced to square fragments of 1mm² and subjected to enzymatic digestion by incubation in RPMI1640medium containing collagenase (1,000 U/ml), hyaluronidase (300 U/ml),and DNase I (100 μg/ml) at 37° C. for 2 h. Primary breast tumor cellswere collected after filtration through a 100-μm cell strainer (BDBiosciences) and resuspended in RPMI1640 medium supplemented with 5%FBS.

Example 14 Flow Cytometry Analysis

Primary breast cancer cells were prepared as 1×10⁵ cells in 50 μl of PBScontaining 2% FBS and 0.1% NaN₃. Cells were labeled with anti-CD24-PE,anti-CD44-APC, and anti-CD45-PerCP-Cy5.5 antibody mixtures (1 μl ofeach). Globo H expression was detected by staining with monoclonalanti-Globo H antibody (VK-9) conjugated with Alexa488. Analyses wereperformed on a FACSCanto flow cytometer (Becton Dickinson). BCSCs weredefined as CD45⁻/CD24⁻/CD44⁺ cells, and non-BCSCs were defined as otherpopulations of CD45 cells. Globo H expression was further analyzed inthe gated region.

Example 15 Cell Sorting

The cells harvested from human breast tumor engrafted in mice werestained with anti-CD24-PE, anti-CD44-APC, and anti-H2K^(d)-FITC antibodymixtures (BD Biosciences). Fluorescence activated sorting ofantibody-labeled cells was carried out on a FACSAria cell sorter (BectonDickinson). H2Kd⁻/CD24⁻/CD44⁺ cells were sorted as BCSCs, and otherpopulations of H2Kd⁻ cells were sorted as non-BCSCs. The typicalpurities of BCSCs and non-BCSCs were >85% and >90%, respectively.

Example 16 Immunohistochemistry

For SSEA-4 expression on normal tissues, tissue microarray slides(Biomax) that contained 20 different organs, with each organ derivedfrom five individuals, were used. Slides were dried overnight at 56° C.,dewaxed in xylene, and rehydrated according to the standardhistopathologic procedures, followed by antigen retrieval with AR-10solution pH 9.0 (BioGenex Laboratories). SSEA-4 expression wasdetermined with the use of anti-SSEA-4 antibody (eBioscience). Stainingfor SSEA-4 was detected by using anti-rat IgM as a secondary antibodyand was developed by DAB substrate. Slides were counterstained withhematoxylin. Primary breast tumor BC0145 and tumor xenografts fromNOD/SCID mice were fixed in 10% phosphate-buffered formalin and embeddedin paraffin. Paraffin sections were cut at a thickness of 2 μM, mountedon SuperFrost Plus microscopy slides (Menzel-Gläser), and driedovernight at 55° C. The sections were dewaxed in xylene and rehydratedaccording to the standard histopathologic procedures, followed bystaining with hematoxylin and eosin (H&E). Before immunostaining, theslides were first placed in the solution of 10 mmol/L citrate buffer (pH6.0) and microwaved for 15 min. The slides were then incubated overnightwith anti-ER, or anti-PR antibody. Immunodetection was performed withthe Super Sensitive Polymer-HRP IHC Detection System (BioGenex).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

The Abstract is provided to comply with 37 C.F.R. §1.72(b) to allow thereader to quickly ascertain the nature and gist of the technicaldisclosure. The Abstract is submitted with the understanding that itwill not be used to interpret or limit the scope or meaning of theclaims.

The invention claimed is:
 1. An immunogenic composition, comprising: (a)one or more glycans comprising Globo H, stage-specific embryonicantigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), Gb4, animmunogenic fragment thereof, or combination thereof, wherein each ofthe glycan is conjugated with a carrier protein selected from diphtheriatoxin cross-reacting material 197 (DT-CRM 197), diphtheria toxoid,tetanus toxoid or bovine serum albumin through a linker; and (b) anα-galactosyl-ceramide (α-GalCer) adjuvant, wherein the adjuvant has thefollowing structure:

wherein the composition induces an antibody that cross-reacts with atleast one of the antigens: Globo H, Gb4, SSEA-3 and SSEA-4.
 2. Theimmunogenic composition of claim 1, wherein the linker is covalentlylinked to the glycan and the carrier protein.
 3. The immunogeniccomposition of claim 1, wherein the linker is a p-nitrophenyl linker. 4.The immunogenic composition of claim 1, further comprising apharmaceutically acceptable excipient.
 5. The immunogenic composition ofclaim 1, wherein the immunogenic composition is capable of eliciting animmune response against a tumor.
 6. The immunogenic composition of claim1, wherein the immunogenic composition induces an immune response thatproduces a higher level of IgG antibody relative to IgM antibody.
 7. Animmunogenic composition, comprising: (a) one or more glycans comprisingSSEA-3, Gb4, an immunogenic fragment thereof, or combination thereof,wherein each of the glycan is conjugated with a carrier protein througha linker; and (b) an adjuvant with the following structure:

wherein the immunogenic composition induces an immune response thatproduces a higher level of IgG antibody relative to IgM antibody.
 8. Theimmunogenic composition of claim 7, wherein the carrier protein is atoxin protein.
 9. The immunogenic composition of claim 8, wherein thecarrier protein is DT-CRM 197, diphtheria toxoid, tetanus toxoid orbovine serum albumin.
 10. The immunogenic composition of claim 7,wherein the linker is covalently linked to the glycan and the carrierprotein.
 11. The immunogenic composition of claim 10, wherein the linkeris a p-nitrophenyl linker.
 12. The immunogenic composition of claim 7,further comprising a pharmaceutically acceptable excipient.
 13. Theimmunogenic composition of claim 1, wherein the glycan is Globo H andthe composition induces the antibody that cross-reacts with SSEA-3. 14.The immunogenic composition of claim 1, wherein the glycan is Globo Hand the composition induces the antibody that cross-reacts with SSEA-4.15. The immunogenic composition of claim 1, wherein the glycan is GloboH and the composition induces the antibody that cross-reacts with Gb-4.